The long-term objective of the proposed research is to establish preclinical efficacy of a platform technology for the stabilization of proteins therapeutics. Inherent instability of protein therapeutics is a large hindrance and in some cases a barrier to their use. We have developed polymers that contain the sugar trehalose in the side chains. The materials stabilize biologics to high temperatures, room temperature, refrigeration, freezing, lyophilization, and even to electron beam sterilization dosages. Furthermore, we have evidence that the polymer confers favorable pharmacokinetic properties to proteins similar to the widely utilized polymer in medicine, poly (ethylene glycol) or PEG. We propose that trehalose glycopolymers will have comparable pharmacokinetic properties to PEG and yet be significantly better than PEG with regard to stabilization ability. This is important for patient health, quality of life, and to reduce costs. We plan to demonstrate this with three important therapeutics: insulin and granulocyte colony-stimulating factor (G-CSF) and hyaluronidase (HAse). These representative proteins have different sizes and degrees of glycosylation and are important to stabilize. Yet, if this research is successful, the polymers may be broadly applicable to many other therapeutics. To reach our major objectives, three specific aims are proposed. First, we will prepare polymer-insulin conjugates and determine bioactivity before and after subjection to heat stress. It is hypothesized that trehalose glycopolymer-protein will be comparable to PEG-protein with regard bioactivity, yet exhibit superior stability to heat compared to the PEG conjugate and unmodified protein. To test this, conjugates will be synthesized and subjected to increases in temperature. The protein integrity will be studied biochemically to evaluate size, aggregation state and folding. The bioactivities will then be determined with standard in vitro assays and in vivo assays in mice for insulin, G-CSF and HAse. Second, we will study pharmacokinetic properties and immune response of polymer conjugates. The hypothesis is that conjugated trehalose glycopolymers will prolong the blood half-life of proteins similar to PEG and will be non antigenic. To determine this, standard pharmacokinetic and immunogenicity studies will be undertaken and compared to the analogous PEGylated versions. Third, we will investigate biodistribution and toxicity of conjugates. It is hypothesized that conjugated trehalose glycopolymers will be nontoxic in vivo and will be distributed and eliminated from mice similarly to PEG. This will be ascertained by biodistribution studies utilizing positron emission tomography and gamma counting. The acute and chronic toxicity will be studied in mice by hematological analysis, biochemical parameters, and histological evaluation. These studies are critical to evaluate if trehalose glycopolymers are safe and can be explored further to enhance the life-times of proteins both inside and outside of the body. Studies to identify polymers that can replace PEG in conjugates are important and are part of our long-term goal to replace PEG in protein conjugates.